Identification of the Maturation Factor for Dual Oxidase

Abstract

Dual oxidase 2 (DUOX2), an NADPH:O 2 oxidoreductase flavoprotein, is a component of the thyroid H 2 O 2 generator crucial for hormone synthesis at the apical membrane. Mutations in DUOX2 produce congenital hypothyroidism in humans. However, no functional DUOX-based NADPH oxidase has ever been reconstituted at the plasma membrane of transfected cells. It has been proposed that DUOX retention in the endo-plasmatic reticulum (ER) of heterologous systems is due to the lack of an unidentified component required for functional maturation of the enzyme. By data mining of a massively parallel signature sequencing tissue expression data base, we identified an uncharacterized gene named DUOX maturation factor (DUOXA2) arranged head-to-head to and co-expressed with DUOX2. A paralog (DUOXA1) was similarly linked to DUOX1. The genomic rearrangement leading to linkage of ancient DUOX and DUOXA genes could be traced back before the divergence of echino-derms. We demonstrate that co-expression of DUOXA2, an ER-resident transmembrane protein, allows ER-to-Golgi transition, maturation, and translocation to the plasma membrane of functional DUOX2 in a heterol-ogous system. The identification of DUOXA genes has important implications for studies of the molecular mechanisms controlling DUOX expression and the molecular genetics of congenital hypothyroidism. Generation of H 2 O 2 at the apical membrane of thyroid follicular cells is essential for iodination of thyroglobulin by thyroid peroxidase and constitutes the rate-limiting step of thyroid hormone synthesis. Dual oxidases (DUOX1 and DUOX2) 2 appear to constitute the catalytic core of the H 2 O 2 generator (1, 2). They are large homologs of the phagocyte gp91 phox /Nox2 NADPH-dependent oxidase with an N-terminal extension comprising a peroxidase-like domain. Although the crucial role of DUOX2 in thyroid hormonogenesis has been substantiated by reports of severe congenital hypothyroidism in patients with biallelic nonsense mutations (3), the understanding of structure, function, and regulation of DUOX has remained limited. The major obstacle for molecular studies of DUOX is the lack of a suitable heterologous cell system for DUOX-based functional NADPH oxidase expression. Transfected cells completely retain DUOX in the endoplasmatic reticulum (ER) (4-8), suggesting that an unidentified component, essential for DUOX maturation, may be specifically expressed in tissues containing the functional enzyme. EXPERIMENTAL PROCEDURES Data Mining and Computational Analysis-Massively parallel signature sequencing (MPSS) data (9) were obtained from the NCBI Gene Expression Omnibus repository (www.ncbi.nlm.nih.gov/geo/; records GSE1747 and GPL1443). A thyroid specificity score, as defined by Jongeneel et al. (9), was calculated for signatures with frequency 100 tags per million (30 mRNA copies/cell) in the thyroid/parathyroid library. Tags with scores 1 were mapped to the human genome assembly using BLAST. DUOXA homologs were identified by tBLASTn searches against the NCBI nr data base and trace archive and BLAT queries (at genome.ucsc.edu/) against assembled whole genome sequences. Orthologs were operationally defined as reciprocal best BLAST hits. Gene structures were deduced by spliced alignment maintaining maximum homolog similarity of the open reading frames (ORFs) and consensus splice junctions. Cla-dograms were constructed from ClustalX alignments (BLOSUM weight matrix, excluding gaps) using the Jones, Taylor, and Thornton (JTT) substitution model in PHYML 2.4.4 (10). SignalP 3.0 (11) and Phobius (12) were used to analyze signal peptides, transmembrane helices, and topology. Northern Blot Analysis-A human multiple tissue Northern blot (Origene) was hybridized with DUOXA2 (125-470 of DQ489734) and DUOXA1 (1244-1623 of BC020841) probes. Heterologous Expression of DUOX2 and DUOXA2 Constructs-cDNA was synthesized with Superscript reverse transcriptase (Invitrogen) by oligo(dT) priming of total RNA from a normal human thyroid gland. The DUOX2 and DUOXA2 ORFs were amplified using native Pfu polymerase (Stratagene) and cloned into pcDNA3.1 (Invitrogen). Epitope-tagged constructs and fusions with enhanced green fluorescent protein (EGFP) were prepared by replacement or splicing-by-overlap extension using specifically designed primers. All constructs were verified by sequencing. HeLa cells were cultured and trans-fected as described (13). Confocal Laser Scanning Microscopy-Indirect immunofluorescence of permeabilized cells has been described previously (13). For surface staining, cells were incubated with rat anti-HA clone 3F10 and/or mouse anti-c-myc clone 9E10 (both from Roche Applied Science) at 1 g/ml in Hank's buffered saline solution/10 mM Hepes, pH 7.4, 1% bovine serum albumin at 4 °C. Rabbit anti-calnexin was obtained from StressGen. Images were captured on a Nikon Eclipse E800 equipped with PCM2000. Analysis of N-Glycosylation-Postnuclear supernatants (in 50 mM Tris/ HCl, pH 8.0, 150 mM NaCl, and proteinase inhibitors) were adjusted to 0.5% SDS, 0.4 mM dithiotreitol and denatured, at room temperature, for 30 min. Samples were deglycosylated with N-glycosidase F (PNGase F) and endogly-cosidase H (Endo H) (both from New England Biolabs) according to manufac-turer's recommendations, followed by SDS-PAGE under reducing conditions and Western blotting as described (13). Measurement of H 2 O 2 Generation-Release of H 2 O 2 was determined by reaction with cell-impermeable 10-acetyl-3,7-dihydroxyphenoxazine (14) (Amplex Red reagent, Invitrogen) in the presence of excess peroxidase, producing fluorescent resorufin. Briefly, cell monolayers were incubated, with or without 10 M diphenyleneiodonium (DPI), in Dulbecco's phosphate-buffered saline supplemented with 50 M Amplex Red reagent and 0.1 unit/ml horseradish peroxidase for 1 h at 37 °C. Relative fluorescence units (excitation/ emission: 535/595) were corrected for Amplex Red oxidation in wells containing non-transfected cells and converted into H 2 O 2 concentrations using a calibration curve. Renilla luciferase activity from co-transfected pRL-Tk plas-mid (Promega) was used as internal control as described (13). RESULTS AND DISCUSSION Identification of Novel Genes in the DUOX1/DUOX2 Intergenic Region-We used MPSS data for 32 normal human tissues (9) to identify novel transcripts with predominant expression in thyroid gland. One of the extracted tags mapped to an uncharacterized locus (LOC405753) oriented head-to-head to DUOX2 in the 16-kbp DUOX1/DUOX2 intergenic region. For reasons outlined below, we called the corresponding gene DUOX maturation factor 2 (DUOXA2). 3 Based on human-mouse homology (Riken clone 9030623N16Rik), and supported by contig assembly of expressed sequence tags (ESTs), it comprises six